Erythropoiesis, the production of red blood cells, occurs continuously throughout the human life span as a compensation for cell destruction. Erythropoiesis is a precisely controlled physiological mechanism enabling sufficient numbers of red blood cells to be available in the blood for proper tissue oxygenation, but not so many that the cells would impede circulation. The maturation of red blood cells is under the control of the hormone, erythropoietin (EPO).
Erythropoietin is an acidic glycoprotein hormone of approximately 34,000 daltons. Naturally occurring erythropoietin is produced by the liver during fetal life and by the kidney in response to hypoxia (e.g., red blood cell loss due to anemia) and regulates red blood cell growth and differentiation through interaction with its cognate cellular receptor cells into erythrocytes. It is essential for regulating levels of red blood cells in blood circulation of adults and stimulates the production of red blood cells in bone marrow. Anemia is a consequence of renal failure to produce erythropoietin. Recombinant erythropoietin produced by genetic engineering techniques involving the expression of a protein product from a host cell transformed with the gene encoding erythropoietin has been found to be effective when used in the treatment of anemia resulting from chronic renal failure. Wild type, or naturally-occurring, erythropoietin is defined herein to include recombinant erythropoietin (Jacobs, K, et al., Nature, 313:806–813 (1985)), or naturally-occurring erythropoietin which has been isolated and purified from blood (Miyake, T., et al., J. Biol. Chem., 252:5558–5564 (1977)) or sheep plasma (Goldwasser, E., et al., Proc. Natl. Acad. Sci. U.S.A., 68:697–698 (1971)), or chemically synthesized erythropoietin which can be produced using techniques well known to those of skill in the art. Human erythropoietin is a 166 amino acid polypeptide that exists naturally as a monomer (Lin, F K., et al., Proc. Natl. Acad. Sci. USA 82:7580–7584 (1985)). The tertiary structure of erythropoietin as an isolated protein and in a complex with its receptor has been reported (Syed R S, et al., Nature [1998] 395:511–6; Cheetham J C, Nat Struct Biol. [1998] 5:861–6). The identification, cloning, and expression of genes encoding erythropoietin are described in U.S. Pat. No. 4,703,008. A description of the purification of recombinant erythropoietin from cell medium that supported the growth of mammalian cells containing recombinant erythropoietin plasmids for example, is included in U.S. Pat. No. 4,667,016. The expression and recovery of biologically active recombinant erythropoietin from a mammalian cell containing the erythropoietin gene on a recombinant plasmid has made available quantities of erythropoietin suitable for therapeutic applications. In addition, knowledge of the gene sequence and the availability of larger quantities of purified protein has led to a better understanding of the mode of action of this protein. Several forms of anemia, including those associated with renal failure, HIV infection, blood loss and chronic disease can be treated with this hematopoietic growth factor. Erythropoietin is typically administered by intravenous or subcutaneous injection three times weekly at a dose of approximately 25–100 U/kg.
Unlike proteins from prokaryotic cells, many cell surface and secretory proteins produced by eukaryotic cells are modified with one or more oligosaccharide groups. This modification, referred to as glycosylation, can dramatically affect the physical properties of proteins and can also be important in protein stability, pharmacokinetics, secretion, and subcellular localization. Proper glycosylation can be essential for biological activity. In fact, some genes from eukaryotic organisms, when expressed in bacteria (e.g., E. coli) which lack cellular processes for glycosylating proteins, yield proteins that are recovered with little or no activity by virtue of their lack of glycosylation. Glycosylation occurs at specific locations along the polypeptide backbone and is usually of two types: 0-linked oligosaccharides are attached to serine or threonine residues while N-linked oligosaccharides are attached to asparagine residues when they are part of the sequence Asn-X-Ser/Thr, where X can be any amino acid except proline. The structures of N-linked and 0-linked oligosaccharides and the sugar residues found in each type are different. One type of sugar that is commonly found on both is N-acetylneuraminic acid (hereafter referred to as sialic acid). Sialic acid is usually the terminal residue of both N-linked and 0-linked oligosaccharides and, by virtue of its negative charge, may confer acidic properties to the glycoprotein. Human recombinant erythropoietin (expressed in mammalian cells) contains three N-linked and one 0-linked oligosaccharide chains which together comprise about 40% of the total molecular weight of the glycoprotein. N-linked glycosylation occurs at asparagine residues (Asn) located at positions 24, 38 and 83 while 0-linked glycosylation occurs at a serine residue (Ser) located at position 126 (Lai et al. J. Biol. Chem. 261, 3116 (1986); Broudy et al. Arch. Biochem. Biophys. 265, 329 (1988)). The oligosaccharide chains have been shown to be modified with terminal sialic acid residues. EPO isoforms having a modified sialic acid pattern are disclosed e.g. in EP 0668 351 or EP 0428 267.
Glycosylation does not seem to be essential for activity, because enzymatically deglycosylated erythropoietin has an activity similar to that of the normally glycosylated protein. However, when the glycosylation sites in erythropoietin are mutated to prevent glycosylation, there is a profound inhibition of the normal synthesis and export of the protein (Dube et al., JBC [1988] 263:17516). Specifically, elimination of glycosylation at Asn38 causes a 99% synthesis block, and elimination of glycosylation at Asn83 causes at least a 99.99% synthesis block, and elimination of glycosylation at Ser126 causes a 99.8% synthesis block.
One problem with erythropoietin therapy is that, although quite effective, this form of therapy is very expensive. Another problem encountered in the practice of medicine when using injectable pharmaceuticals is the frequency at which those injections must be made in order to maintain a therapeutic level of the compound in the circulation. For example, erythropoietin has a relatively short plasma half-life (Spivak, J. L., and Hogans, B. B., Blood, 73:90 (1989); McMahon, F. G., et al., Blood, 76:1718(1990)), therefore, therapeutic plasma levels are rapidly lost, and repeated intravenous administrations must be made.
It would be advantageous to have available derivatives of erythropoietin which have an extended circulating half-life to avoid such problems. In addition one would prefer to synthesize EPO in a host cell other than a mammalian cell. Unfortunately, synthesis in bacteria is problematic because the protein is not produced in a properly folded, native conformation. Synthesis in insect cells or plant cells is also problematic because these cells provide an unfavorable glycosylation pattern. Proteins that are glycosylated according to the insect pattern or the plant patterns are, upon injection into animals, generally taken up by specific receptors and rapidly degraded. For example, macrophages in the liver possess high mannose receptors and asialo-glycoprotein receptors that remove proteins with non-mammalian glycosylation patterns.